MATERIAL AND METHOD: The purity of mitragynine in a Mitragyna speciosa alkaloid extract (MSAE) was determined using Ultra-Fast Liquid Chromatography (UFLC). In vitro high throughput ADMETox studies such as aqueous solubility, plasma protein binding, metabolic stability, permeability and cytotoxicity tests were carried out to analyze the physicochemical properties of MSAE and mitragynine. The UFLC quantification revealed that the purity of mitragynine in the MSAE was 40.9%.

RESULTS: MSAE and mitragynine are highly soluble in aqueous solution at pH 4.0 but less soluble at pH 7.4. A parallel artificial membrane permeability assay demonstrated that it is extensively absorbed through the semi-permeable membrane at pH 7.4 but very poorly at pH 4.0. Both are relatively highly bound to plasma proteins (> 85 % bound) and are metabolically stable to liver microsomes (> 84 % remained unchanged). In comparison to MSAE, mitragynine showed higher cytotoxicity against WRL 68, HepG2 and Clone 9 hepatocytes after 72 h treatment.

METHODS: This study investigated the microbial composition and readily found bioactive compounds in water kefir fermented in Malaysia using 16S rRNA microbiome and UHPLC sequencing approaches. The toxicity effects of the kefir water administration in BALB/c mice were analysed based on the mice survival, body weight index, biochemistry profile, and histopathological changes. The antioxidant activities were evaluated using SOD, FRAP, and NO assays.

RESULTS: The 16S rRNA amplicon sequencing revealed the most abundant species found in the water kefir was Lactobacillus hilgardii followed by Lactobacillus harbinensis, Acetobacter lovaniensis, Lactobacillus satsumensis, Acetobacter tropicalis, Lactobacillus zeae, and Oenococcus oeni. The UHPLC screening showed flavonoid and phenolic acid derivatives as the most important bioactive compounds present in kefir water which has been responsible for its antioxidant activities. Subchronic toxicity study showed no toxicological signs, behavioural changes, or adverse effects by administrating 10 mL/kg/day and 2.5 mL/kg/day kefir water to the mice. Antioxidants assays demonstrated enhanced SOD and FRAP activities and reduced NO level, especially in the brain and kidney samples.

METHOD: Antioxidant activities were determined. Phytochemical analysis was performed by gas chromatography mass spectrometry (GCMS). In the in vivo study, Sprague Dawley rats were pretreated with C. nudiflora (150, 300, and 450 mg kg body weight (b.wt.)) once daily for 14 days followed by two doses of CCl4 (1 ml/kg b.wt.). After 2 weeks, the rats were sacrificed and hepatoprotective analysis was performed.

RESULTS: In vitro studies have shown that the extract possessed strong antioxidant activity and has ability to scavenge 2,2-diphenyl-2-picrylhydrazyl-free radicals effectively. GCMS analysis of the C. nudiflora extract revealed the presence of various bioactive compounds. Administration of C. nudiflora significantly reduced the impact of CCl4 toxicity on serum markers of liver damage, serum aspartate transaminase (AST), and alanine transaminase (ALT). C. nudiflora also increased antioxidant levels of hepatic glutathione (GSH) and antioxidant enzymes and ameliorated the elevated hepatic formation of malondialdehyde (MDA) induced by CCl4 in rats. Histopathological examination indicated that C. nudiflora protect the liver from the toxic effect of CCl4 and healed lesions such as necrosis, fatty degeneration, and hepatocyte injury as irregular lamellar organization and dilations in the endoplasmic reticulum. The immunohistochemical studies revealed that pretreatment of C. nudiflora decreased the formation of 4-hydroxy-2-nonenal (HNE)-modified protein adducts and 8-hydroxy-2'-deoxyguanosine (8-OHdG). Furthermore, overexpression of the proinflammatory cytokines TNF-α, IL-6, and prostaglandin E2 is also reduced.